The present invention relates to vertebrate Delta genes and their encoded protein products, as well as derivatives and analogs thereof. Production of vertebrate Delta proteins, derivatives, and antibodies is also provided. The invention further relates to therapeutic compositions and methods of diagnosis and therapy.
Genetic analyses in Drosophila have been extremely useful in dissecting the complexity of developmental pathways and identifying interacting loci. However, understanding the precise nature of the processes that underlie genetic interactions requires a knowledge of the protein products of the genes in question.
The vertebrate central nervous system is an intimate mixture of different cell types, almost all generated from the same sourcexe2x80x94the neurogenic epithelium that forms the neural plate and subsequently the neural tube. What are the mechanisms that control neurogenesis in this sheet of cells, directing some to become neurons while others remain non-neuronal? The answer is virtually unknown for vertebrates, but many of the cellular interactions and genes controlling cell fate decisions during neurogenesis have been well characterized in Drosophila (Campos-Ortega, 1993, J. Neurobiol. 24:1305-1327). Although the gross anatomical context of neurogenesis appears very different in insects and vertebrates, the possibility remains that, at a cellular level, similar events are occurring via conserved molecular mechanisms. Embryological, genetic and molecular evidence indicates that the early steps of ectodermal differentiation in Drosophila depend on cell interactions (Doe and Goodman, 1985, Dev. Biol. 111:206-219; Technau and Campos-Ortega, 1986, Dev. Biol. 195:445-454; Vxc3xa4ssin et al., 1985, J. Neurogenet. 2:291-308; de la Concha et al., 1988, Genetics 118:499-508; Xu et al., 1990, Genes Dev. 4:464-475; Artavanis-Tsakonas, 1988, Trends Genet. 4:95-100). Mutational analyses reveal a small group of zygotically-acting genes, the so called neurogenic loci, which affect the choice of ectodermal cells between epidermal and neural pathways (Poulson, 1937, Proc. Natl. Acad. Sci. 23:133-137; Lehmann et al., 1983, Wilhelm Roux""s Arch. Dev. Biol. 192:62-74; Jxc3xcrgens et al., 1984, Wilhelm Roux""s Arch. Dev. Biol. 193:283-295; Wieschaus et al., 1984, Wilhelm Roux""s Arch. Dev. Biol. 193:296-307; Nxc3xcsslein-Volhard et al., 1984, Wilhelm Roux""s Arch. Dev. Biol. 193:267-282). Null mutations in any one of the zygotic neurogenic locixe2x80x94Notch (N), Delta (Dl), mastermind (mam), Enhancer of Split (E(spl), neuralized (neu), and big brain (bib)xe2x80x94result in hypertrophy of the nervous system at the expense of ventral and lateral epidermal structures. This effect is due to the misrouting of epidermal precursor cells into a neuronal pathway, and implies that neurogenic gene function is necessary to divert cells within the neurogenic region from a neuronal fate to an epithelial fate.
Neural precursors arise in the Drosophila embryo from a neurogenic epithelium during successive waves of neurogenesis (Campos-Ortega and Hartenstein, 1985, The embryonic development of Drosophila melanogaster (Springer-Verlag, Berlin; New York); Doe, 1992, Development 116:855-863). The pattern of production of these cells is largely determined by the activity of the proneural and neurogenic genes. Proneural genes predispose clusters of cells to a neural fate (reviewed in Skeath and Carroll, 1994, Faseb J. 8:714-21), but only a subset of cells in a cluster become neural precursors. This restriction is due to the action of the neurogenic genes, which mediate lateral inhibitionxe2x80x94a type of inhibitory cell signaling by which a cell committed to a neural fate forces its neighbors either to remain uncommitted or to enter a non-neural pathway (Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408; Doe and Goodman, 1985, Dev. Biol. 111:206-219). Mutations leading to a failure of lateral inhibition cause an overproduction of neuronsxe2x80x94the xe2x80x9cneurogenicxe2x80x9d phenotype (Lehmann et al., 1981, Roux""s Arch. Dev. Biol. 190:226-229; Lehmann et al., Roux""s Arch. Dev. Biol. 192:62-74). In Drosophila, the inhibitory signal is delivered by a transmembrane protein encoded by the Delta neurogenic gene, which is displayed by the nascent neural cells (Heitzler and Simpson, 1991, Cell 64:1083-1092). Neighboring cells express a transmembrane receptor protein, encoded by the neurogenic gene Notch (Fortini and Artavanis-Tsakonas, 1993, Cell 75:1245-1247). Delta has been identified as a genetic unit capable of interacting with the Notch locus (Xu et al., 1990, Genes Dev. 4:464-475).
Mutational analyses also reveal that the action of the neurogenic genes is pleiotropic and is not limited solely to embryogenesis. For example, ommatidial, bristle and wing formation, which are known also to depend upon cell interactions, are affected by neurogenic mutations (Morgan et al., 1925, Bibliogr. Genet. 2:1-226; Welshons, 1956, Dros. Inf. Serv. 30:157-158; Preiss et al., 1988, EMBO J. 7:3917-3927; Shellenbarger and Mohler, 1978, Dev. Biol. 62:432-446; Technau and Campos-Ortega, 1986, Wilhelm Roux""s Dev. Biol. 195:445-454; Tomlison and Ready, 1987, Dev. Biol. 120:366-376; Cagan and Ready, 1989, Genes Dev. 3:1099-1112). Neurogenic genes are also required for normal development of the muscles, gut, excretory and reproductive systems of the fly (Muskavitch, 1994, Dev. Biol. 166:415-430).
Both Notch and Delta are transmembrane proteins that span the membrane a single time (Wharton et al., 1985, Cell 43:567-581; Kidd and Young, 1986, Mol. Cell. Biol. 6:3094-3108; Vxc3xa4ssin, et al., 1987, EMBO J. 6:3431-3440; Kopczynski, et al., 1988, Genes Dev. 2:1723-1735) and include multiple tandem EGF-like repeats in their extracellular domains (Muskavitch, 1994, Dev. Biol. 166:415-430). The Notch gene encodes a xcx9c300 kd protein (we use xe2x80x9cNotchxe2x80x9d to denote this protein) with a large N-terminal extracellular domain that includes 36 epidermal growth factor (EGF)-like tandem repeats followed by three other cysteine-rich repeats, designated Notch/lin-12 repeats (Wharton, et al., 1985, Cell 43:567-581; Kidd and Young, 1986, Mol. Cell. Biol. 6:3094-3108; Yochem, et al., 1988, Nature 335:547-550). Molecular studies have lead to the suggestion that Notch and Delta constitute biochemically interacting elements of a cell communication mechanism involved in early developmental decisions (Fehon et al., 1990, Cell 61:523-534). Homologs are found in Caenorhabditis elegans, where the Notch-related gene lin-12 and the Delta-related gene lag-2 are also responsible for lateral inhibition (Sternberg, 1993, Current Biol. 3:763-765; Henderson et al., 1994, Development 120:2913-2924; Greenwald, 1994, Curr. Opin. Genet. Dev. 4:556-562). In vertebrates, several Notch homologs have also been identified (Kopan and Weintraub, 1993, J. Cell Biol. 121:631-641; Lardelli et al., 1994, Mech. Dev. 46:123-136; Lardelli and Lendahl, 1993, Exp. Cell Res. 204:364-372; Weinmaster et al., 1991, Development 113:199-205; Weimnaster et al., 1992, Development 116:931-941; Coffman et al., 1990, Science 249:1438-1441; Bierkamp and Campos-Ortega, 1993, Mech. Dev. 43:87-100), and they are expressed in many tissues and at many stages of development. Loss of Notch-1 leads to somite defects and embryonic death in mice (Swiatek et al., 1994, Genes Dev. 8:707-719; Conlon et al., Rossant, J. Development (J. Dev. 121:1533-1545), while constitutively active mutant forms of Notch-1 appear to inhibit cell differentiation in Xenopus and in cultured mammalian cells (Coffman et al., 1993, Cell 73:659-671; Kopan et al., 1994, Development 120:2385-2396; Nye et al., 1994, Development 120:2421-2430).
The EGF-like motif has been found in a variety of proteins, including those involved in the blood clotting cascade (Furie and Furie, 1988, Cell 53: 505-518). In particular, this motif has been found in extracellular proteins such as the blood clotting factors IX and X (Rees et al., 1988, EMBO J. 7:2053-2061; Furie and Furie, 1988, Cell 53: 505-518), in other Drosophila genes (Knust et al., 1987 EMBO J. 761-766; Rothberg et al., 1988, Cell 55:1047-1059), and in some cell-surface receptor proteins, such as thrombomodulin (Suzuki et al., 1987, EMBO J. 6:1891-1897) and LDL receptor (Sudhof et al., 1985, Science 228:815-822). A protein binding site has been mapped to the EGF repeat domain in thrombomodulin and urokinase (Kurosawa et al., 1988, J. Biol. Chem 263:5993-5996; Appella et al., 1987, J. Biol. Chem. 262:4437-4440).
Citation of references hereinabove shall not be construed as an admission that such references are prior art to the present invention.
The present invention relates to nucleotide sequences of vertebrate Delta genes (chick and mouse Delta, and related genes of other species), and amino acid sequences of their encoded proteins, as well as derivatives (e.g., fragments) and analogs thereof. Nucleic acids hybridizable to or complementary to the foregoing nucleotide sequences are also provided. In a specific embodiment, the Delta protein is a mammalian protein, preferably a human protein.
The invention relates to vertebrate Delta derivatives and analogs of the invention which are functionally active, i.e., they are capable of displaying one or more known functional activities associated with a full-length (wild-type) Delta protein. Such functional activities include but are not limited to antigenicity [ability to bind (or compete with Delta for binding) to an anti-Delta antibody], immunogenicity (ability to generate antibody which binds to Delta), ability to bind (or compete with Delta for binding) to Notch or other toporythmic proteins or fragments thereof (xe2x80x9cadhesivenessxe2x80x9d), ability to bind (or compete with Delta for binding) to a receptor for Delta. xe2x80x9cToporythmic proteinsxe2x80x9d as used herein, refers to the protein products of Notch, Delta, Serrate, Enhancer of split, and Deltex, as well as other members of this interacting set of genes which may be identified, e.g., by virtue of the ability of their gene sequences to hybridize, or their homology to Delta, Serrate, or Notch, or the ability of their genes to display phenotypic interactions or the ability of their protein products to interact biochemically.
The invention further relates to fragments (and derivatives and analogs thereof) of a vertebrate Delta that comprise one or more domains of the Delta protein, including but not limited to the intracellular domain, extracellular domain, transmembrane domain, DSL domain, domain amino-terminal to the DSL domain, or one or more EGF-like (homologous) repeats of a Delta protein, or any combination of the foregoing.
Antibodies to a vertebrate Delta, its derivatives and analogs, are additionally provided.
Methods of production of the vertebrate Delta proteins, derivatives and analogs, e.g., by recombinant means, are also provided.
The present invention also relates to therapeutic and diagnostic methods and compositions based on Delta proteins and nucleic acids. The invention provides for treatment of disorders of cell fate or differentiation by administration of a therapeutic compound of the invention. Such therapeutic compounds (termed herein xe2x80x9cTherapeuticsxe2x80x9d) include: Delta proteins and analogs and derivatives (including fragments) thereof; antibodies thereto; nucleic acids encoding the Delta proteins, analogs, or derivatives; and Delta antisense nucleic acids. In a preferred embodiment, a Therapeutic of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non-malignant state into a neoplastic or a malignant state. In other specific embodiments, a Therapeutic of the invention is administered to treat a nervous system disorder or to promote tissue regeneration and repair.
In one embodiment, Therapeutics which antagonize, or inhibit, Notch and/or Delta function (hereinafter xe2x80x9cAntagonist Therapeuticsxe2x80x9d) are administered for therapeutic effect. In another embodiment, Therapeutics which promote Notch and/or Delta function (hereinafter xe2x80x9cAgonist Therapeuticsxe2x80x9d) are administered for therapeutic effect.
Disorders of cell fate, in particular hyperproliferative (e.g., cancer) or hypoproliferative disorders, involving aberrant or undesirable levels of expression or activity or localization of Notch and/or Delta protein can be diagnosed by detecting such levels, as described more fully infra.
In a preferred aspect, a Therapeutic of the invention is a protein consisting of at least a fragment (termed herein xe2x80x9cadhesive fragmentxe2x80x9d) of Delta which mediates binding to a Notch protein or a fragment thereof.
As used herein, underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its encoded protein product which is indicated by the name of the gene in the absence of any underscoring. For example, xe2x80x9cDeltaxe2x80x9d shall mean the Delta gene, whereas xe2x80x9cDeltaxe2x80x9d shall indicate the protein product of the Delta gene.